title: Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella.
abstract: Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of innate immunity. In this study, a long-form PGRP, designated as gcPGRP6, was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP6 is composed of 464 residues with a conserved PGRP domain at the C-terminus. The gcPGRP6 gene consists of four exons and three introns, spacing approximately 2.7 kb of genomic sequence. Phylogenetic analysis demonstrated that gcPGRP6 is clustered closely with zebrafish PGLYRP6, and formed a long-type PGRP subfamily together with PGLYRP2 members identified in teleosts and mammals. Real-time PCR and Western blotting analyses revealed that gcPGRP6 is constitutively expressed in organs/tissues examined, and its expression was significantly induced in liver and intestine of grass carp in response to PGN stimulation and in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and peptidoglycan (PGN). Immunofluorescence microscopy and Western blotting analyses revealed that gcPGRP6 is effectively secreted to the exterior of CIK cells. The over-expression of gcPGRP6 in CIK cells leads to the activation of NF-κB and the inhibition of intracellular bacterial growth. Moreover, cell lysates from CIK cells transfected with pTurbo-gcPGRP6-GFP plasmid display the binding activity towards Lys-type PGN from Staphylococcus aureus and DAP-type PGN from Bacillus subtilis. Furthermore, proinflammatory cytokine IL-2 and intracellular PGN receptor NOD2 had a significantly increased expression in CIK cells overexpressed with gcPGRP6. It is demonstrated that the PGRP6 in grass carp has a role in binding PGN, in inhibiting the growth of intracellular bacteria, and in activating NF-κB, as well as in regulating innate immune genes.
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, China; Graduate University of Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Jun Hua
authors.initials: JH
authors.lastname: Li
authors.forename: Zhang Long
authors.initials: ZL
authors.lastname: Yu
authors.forename: Na Na
authors.initials: NN
authors.lastname: Xue
authors.forename: Peng Fei
authors.initials: PF
authors.lastname: Zou
authors.forename: Jing Yu
authors.initials: JY
authors.lastname: Hu
authors.forename: P
authors.initials: P
authors.lastname: Nie
authors.forename: Ming Xian
authors.initials: MX
authors.lastname: Chang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000276:immunology; D001483:Base Sequence; D002347:Carps / Q000235:genetics / Q000276:immunology*; D002352:Carrier Proteins / Q000235:genetics* / Q000276:immunology* / Q000493:pharmacokinetics; D002460:Cell Line; D003001:Cloning, Molecular; D020609:Edwardsiella tarda / Q000276:immunology; D004756:Enterobacteriaceae Infections / Q000276:immunology*; D029941:Fish Proteins / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis / Q000276:immunology; D007422:Intestines / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D008099:Liver / Q000276:immunology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000276:immunology*; D053473:Nod2 Signaling Adaptor Protein / Q000096:biosynthesis / Q000276:immunology; D010457:Peptidoglycan / Q000276:immunology; D010802:Phylogeny; D011070:Poly I-C / Q000276:immunology; D011485:Protein Binding; D013211:Staphylococcus aureus / Q000276:immunology; D013682:Teichoic Acids / Q000276:immunology
chemical_list: D002352:Carrier Proteins; D029941:Fish Proteins; D007376:Interleukin-2; D008070:Lipopolysaccharides; D016328:NF-kappa B; D053473:Nod2 Signaling Adaptor Protein; D010457:Peptidoglycan; D013682:Teichoic Acids; C118897:peptidoglycan recognition protein; C009900:lipoteichoic acid; D011070:Poly I-C
keywords: Antibacterial activity; Ctenopharyngodon idella; Grass carp; PGRP6; Peptidoglycan recognition protein; Peptidoglycan-binding activity